The purpose of this proposal is to elucidate changes in ionic conditions in the mitotic apparatus (MA) that accompany and perhaps induce the initial separation of chromosomes at the metaphase/anaphase transition. Changes in intracellular ion levels during mitosis can be observed in living cells (e.g., spider spermatocytes, spiderwort stamen hairs or blood lilly endosperm) by spectrophotometry or fluorometry with the light microscope using metallochromic dyes, which are sensitive to specific ions (e.g., H+, Ca2+). Ca2+, an ion whose concentration fluctuations have been found to play a major role as a "second messenger" in many types of eucaryotic cells, may activate chromosome motion. Free Ca2+ concentrations in the MA con be monitored in three ways: a) with Ca2+ specific microelectrodes, b) microspectrophotometrically using Arsenazo III, and c) microflurometrically using BAPTA or Quin-2. Perturbations of intracellular levels of Ca2+ by treatment with the Caionophore A23187, and chelators such as EGTA will be used to assess the effects of altered [Ca2+] on mitotic events. In concert with, or as a consequence of Ca2+ fluctuations, changes in intracellular pHi(pHi) may accompany chromosome movement. Detection of changes in pH by treatment with acetate, NH or nigericin will similarly be used to judge both the effects of altered pH on mitotic events and the methods of measurement. These studies on Ca2+ and H+ will further augment our understanding of the regulation of mitosis, a process whose operation is integral to normal development throughout the eukaryotic realm.